Method and composition for a protein transduction technology and its applications

ABSTRACT

A protein transduction method for efficiently delivery of exogenous proteins into mammalian cells is invented, which has the capability of targeting different cellular compartments and protection from degradation of the delivered proteins from cellular proteases. A composition for treat proteins has cation reagents, lipids and enhancers in a carrier. The method can be used in a number of ways including: production of large quantities of properly folded, post-translationally modified proteins using mammalian cell machinery, a in-cell fluorescence spectroscopy and imaging using small molecule fluorophores and a in-cell NMR spectroscopy using living mammalian cells. The method permits cell biology at atomic resolution that is physiologically and pathological relevant and permits protein therapy to treat human diseases. The method can also be used to deliver exogenous protein inside mammalian cells, wherein the exogenous proteins follow a similar secretion pathway as that of the endogenous protein.

REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.12/128,320, filed May 28, 2008, the entire content of which isincorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention relates to protein transduction and uses thereof.More specifically, the present invention relates to protein transductionreagents that enable proteins to be delivered into mammalian cells andhow to uses of the protein transduction technology, including in vivoprotein folding, trafficking, secretion pathways of transduced exogenousproteins, production of large quantities of native folded,post-translationally modified proteins, in-cell structural biology andprotein therapy.

2. Description of the Related Art

Proteins are necklaces of amino acids, long chain molecules. Proteinsare the most important molecules inside every living cell, tissue, organwithin the human body. Proteins are involved in virtually all aspects oflife. Proteins control thinking, they regulate all physiologicalreactions, they metabolize carbohydrates and fats that bodies use, theydefend bodies against bacteria and viruses, and they work as enzymes,hormones, antibodies, cytokines and signaling molecules that transmitinformation into cells. As enzymes, they are the driving force behindall of the biochemical reactions. As structural elements, they are themain constituent of bones, muscles, hair, skin and blood vessels. Asantibodies, they recognize invading elements and allow the immune systemto eliminate the unwanted invaders. While scientists have sequenced thehuman genome, how proteins work largely remains a mystery. This isbecause in order for proteins to function (e.g. as enzymes orantibodies), the protein must take on a particular shape, also known asa “fold”. If the protein does not fold correctly, disease anddysfunction occur. Some examples of which include, but are not limitedto, Alzheimer's disease, Huntington's disease, cystic fibrosis, BSE (MadCow disease), an inherited form of emphysema, and even many cancers.When proteins misfold, they can clump together (“aggregate”). Theseclumps can often gather in the brain, where they are believed to causethe symptoms of Mad Cow or Alzheimer's disease.

When proteins fold inside a cell, they are frequently subjected tovarious amounts of spatial confinement. In many cases, proteins arefolded in the endoplasmic reticulum (“ER”), which is amembrane-containing cellular compartment that contains many proteins atspecific concentrations. Proteins can be encapsulated inside helpermolecules, called chaperones and folding, enzymes. These chaperones areinvolved with helping proteins fold inside cells. Therefore, proteinfolding inside the cell is quite different from its folding in the testtube. However, current studies of protein folding are mainly in the testtube. Although significant advance has been achieved, these results ofprotein folding in the test tube have to be verified in living cells andsuch a technique of study protein folding in the living cell is lacking.

The study of protein structure has received a major boost recently withthe increasing amount of structures being deposited on the Protein DataBank (PDB) on a daily basis, but these structures are typically notdetermined in viva, but in artificial crystals and solutions. Over thepast five decades, X-ray crystallography and the resulting atomic modelsof proteins and nucleic acids have contributed greatly to anunderstanding of structural, molecular, and chemical aspects ofbiological phenomena. Currently, X-ray crystallography is a maturehigh-resolution structural biology tool that can be used to quicklydetermine protein structures. However, X-ray crystallography requireshigh quality single crystal of proteins in order to do x-ray diffractionand this is not always achievable. In contrast, another high-resolutionstructural biology tool, nuclear magnetic resonance (NMR), has beendeveloped since 1980s. This technique only requires protein in solutionat 50 μM to 1 mM concentrations. This technique also provides proteindynamics information via NMR relaxation measurements. Although NMR is aless mature high-resolution structural tool, it provides an alternativehigh-resolution structural biology technique, allowing for determinationof protein structures at atomic resolution.

When these methods cannot be used, computer-based protein modelingtechniques have been used with some success. These modeling techniquesuse the known three-dimensional structure of a homologous protein toapproximate the structure of another protein. This is not an accuratemethod because the actual structure is not known, but is approximated.

Fluorescence spectroscopy is another structural technique, which can beused to obtain structural information. The well-developed Forsterresonance energy transfer (FRET) technique can be used to measure thedistance between fluorescence donor and acceptor, and can thus provideimportant information about protein folding and structure. However, FRETcan only provide one distance from one pair of fluorescence donor andacceptor each time. To determine a protein structure, hundreds tothousands of distances within a protein are necessary to determineprotein structure at atomic resolution. This requires enormous amount ofwork, including mutagenesis, protein production, fluorescence labelingand FRET measurement of every pair of fluorescence donor and acceptor.The FRET measurement can also be obtained in the living cells since theintroduction of Green/Red Fluorescence Protein (GFP/RFP) technique. Thein vivo FRET measurement is widely used to study protein-protein anddomain-domain interactions, however, the distance measurement betweenGFP and RFP seems to be meaningless, since both GFP and RFP are proteinsof 25-28 kDa. Thus, the current in vivo FRET measurement cannot be usedto obtain accurate information about protein structure and folding.Therefore, fluorescence spectroscopy is not considered to be a viablehigh-resolution structural biology tool.

Fluorescence imaging is a technique that is routinely used to studyprotein location and trafficking in living cells. Currently, thistechnique extensively utilizes GFP technique, which fuses GFP in eitherthe N- or the C-terminal end of a protein. Using a confocal microscope,the GFP-labeled protein can be visualized for their locations andtrafficking inside the cells. However, it is unknown whether the GFPfusion changes the location of the protein of interest inside the cells.Thus, extensive control experiments have to be carried out. Even withthese control experiments, sometimes the situation inside the cells iscomplex and no definite conclusion can be made using fluorescenceimaging.

Currently, there is no available means for detecting high-resolutionprotein structure in living cells. However, it is critical forscientists to verify if the in vitro determined protein structures arethe same as the structures of these proteins in living cells. An in-cellstructural biology is necessary to push the current cell biology toatomic resolution and no such technology is currently available. Inaddition, this in-cell structural biology will allow us to combine cellbiology techniques with high-resolution structural biology techniques,thus to accurately correlate protein structural information withcellular functions.

Using bacteria to produce recombinant proteins opens the door of modernmolecular biology. Indeed, bacterial expression enables us to utilizethe recombinant DNA technique to produce large quantities of recombinantproteins. When bacterial cells are used to overexpress exogenousproteins, the recombinant protein is often sequestered in bacterial cellinclusion bodies. For the recombinant proteins to be useful, they mustbe purified from the inclusion bodies. During the purification process,the recombinant proteins are denatured and must then be re-natured.Denatured protein is commonly refolded in vitro by diluting thedenaturant away. Protein unfolding normally induces a hydrophobiccollapse that may cause protein aggregation. In vitro protein refoldingresults in the protein shielding its hydrophobic patches in the core ofthe molecule. Unfortunately, during the in vitro refolding process,proteins do not always form the native bioactive conformation. Twocompeting processes occur: refolding and aggregation. It is suggestedthat the driver for protein aggregation is hydrophobic amino acidsexposed at the surface. Aggregation is undesirable and reduces the yieldof functional, native protein.

Bacterial cells cannot be used to produce many proteins due tomisfolding of these proteins in the bacterial cells. In addition,bacterial cells do not contain complex machinery for proteinpost-translational modification. However, protein post-translationalmodifications are critical for the biological functions of manyproteins. Thus, the bacterial expressed recombinant proteins are not thesame as the native proteins and are not functional. For production ofnative proteins, mammalian cells must be used. Unlike bacterialexpression, the yield of mammalian cell protein expression is much lowerand costly. A new technology of production of large quantities ofproperly folded, post-translationally modified proteins is definitelynecessary for modern biology and medical sciences.

The impermeable nature of the cell membrane to peptide, protein, DNA andRNA limits the therapeutic potential of these “information-rich”biological molecules and prevents the uptake of the in vitro labeledmacromolecules by cells for structural biology studies in the livingcell at atomic resolution. However, a new, non-invasive proteintransduction technology is emerging, following the discovery of the cellpenetrating peptide (CPP) that is successfully used to efficientlytransport heterogeneous bioactive cargo into the cell in anunconventional way. The protein transduction technology in vivo todeliver bioactive cargo into various tissues of living animals has beenreported. This novel technology opens up many new possibilities forintracellular delivery of therapeutic macromolecules for treatment ofhuman diseases or for intracellular transduction of labeledmacromolecules for structural biology studies in living cells, thuspotentially pushing cell biology to atomic resolution.

Despite these notable successes, the use of protein transductiontechnology has yet to become commonplace in cell biology and intherapeutic applications. Several major challenges lay in front of thisnew technology that prevent it to be widely used in many fields ofbiomedical sciences. The first challenge is the fate or secretionpathway of delivered exogenous proteins using protein transductiontechnology. It is still unknown how the exogenous proteins trafficinside cells after being delivered into cells. The famous Blobel's“Signal Theory” guides the fate of endogenous protein to traffic insidethe cells, and thus dictates the subcellular locations of endogenousproteins. Questions have arisen regarding whether the exogenous proteinsfollow the same secretion pathway as that of the endogenous proteins.These questions have to be addressed for physiological and pathologicalrelevance of protein transduction technology. The second major challengeis lack of delivery specificity of the current protein transductionagents, specifically, in terms of targeting to specific cell types andspecific cell compartments. Indeed, the current protein transductionreagents are not “smart” enough to specifically deliver exogenousproteins into a targeting tissue type or cellular compartment.

Most human diseases are related to the malfunctioning of particularproteins, either systemically or locally. Therapeutic proteins,including native and engineered proteins, can be used as highlyeffective medical treatments (protein therapy) for a wide array ofdiseases in which the protein is either lacking or deficient (growthhormone and insulin), or the therapeutic protein is used to inhibit abiological process (antibodies that block blood supply to tumors). Incontrast to gene therapy, protein therapy uses well-defined, preciselystructured proteins, with previously defined optimal doses of theindividual protein for disease states, and with well-known biologicaleffects. However, an obstacle currently hinders protein therapy as atreatment of human diseases. This obstacle is the mode of delivery:oral, intravenous, intra-arterial, or intramuscular routes ofadministration are not always as effective as desired. In most cases,the therapeutic protein is metabolized or cleared before it even reachesthe target tissue. To make protein therapy possible, an efficientdelivery system of protein is required to ensure that therapeuticprotein is stable and able to deliver to the target tissues fortreatment of the diseases.

SUMMARY OF THE INVENTION

The present invention is a method for protein transduction intomammalian cells and the applications of this protein transductionmethod. The method can be used for the production of large quantity ofnative folded, post-translationally modified proteins using mammaliancell folding/post-translational modification machinery. The method canalso be used for studies of protein folding, structure, interactions andtrafficking in living mammalian cell, using in-cell fluorescencespectroscopy and imaging and in-cell NMR spectroscopy.

The present invention can also be used to develop the physiologicallyand pathologically relevant, atomic resolution cell biology and proteintherapy to treat human diseases.

The QQ series protein transduction reagent of the present invention canefficiently deliver proteins into mammalian cell, making this methodpossible.

A composition of QQ reagents for treating proteins having cationreagents, lipids, and enhancers in a carrier is provided. Accordingly,the amount of modification of target protein with QQ reagents can beadjusted by altering the compositions to obtain the best proteintransduction efficiency. The composition enable proteins to be deliveredinto mammalian cells and properly fold and post-translationallymodified.

The method of the present invention can also include a labeling step,either NMR or fluorescence labeling, that includes labeling the proteinprior to QQ reagent modification. The labeling enables better viewing ofthe protein within cells using high-resolution biophysical methods.

The method can be used in a number of ways including: production oflarge quantities of properly folded, post-translationally modifiedproteins using mammalian cell machinery, a in-cell fluorescencespectroscopy and imaging using small molecule fluorophores and a in-cellNMR spectroscopy using living mammalian cells. The method permits cellbiology at atomic resolution that is physiologically and pathologicalrelevant and protein therapy to treat human diseases.

The method can also be used to deliver exogenous protein insidemammalian cells, wherein the exogenous proteins follow a similarsecretion pathway as that of the endogenous protein.

These and other objects, advantages and features of the invention willbe more fully understood and appreciated by reference to the descriptionof the current embodiment and the drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow chart depicting the method of the present invention,showing a simple step of utilization of the QQ reagents;

FIGS. 2A and 2B are photographs of western blots using anti-his-tag andanti LBD-apoER2 antibodies, qualitatively showing the efficiency ofprotein transduction into mammalian cells using the present invention;

FIG. 3 is a photograph of a SDS-PAGE, quantitatively showing that thepresent invention can be used to deliver 50-200 μm apoE(1-183) into Helacells;

FIGS. 4A and 4B show photographs of fluorescence imaging of Hela cellsthat were treated with fluorescence labeled, QQ-reagent modifiedLBD-apoER2 and MESD proteins, showing that the delivered proteins wereable to target the ER and the Golgi;

FIGS. 5A and 5B show photographs of SDS-PAGEs, suggesting that the QQreagent modified mouse apoAI was protected from degradation byproteases, both protease cocktail and cellular proteases;

FIG. 5C is a diagram of an experimental procedure;

FIGS. 6A and 6B are photographs of far western blot of LBD-apoER2 usinganti-RAP (FIG. 6A) and anti-apoE (FIG. 6B) antibodies, showing that thetransduced LBD-apoER2 was properly folded and functional for binding toboth. RAP and apoE;

FIG. 7A shows diagrams of MESD proteins;

FIGS. 7B, 7C and 7D are photographs of western blots of the exogenousMESD proteins that were delivered into GM01300 cells (FIG. 7B) and theendogenous MESD from eight different mammalian cell lines (FIG. 7C),showing the same three band pattern. In contrast, MESD(12-155), whichlacks the “REDL” ER retention signal, only shows one single band that isthe same as the bacterial expressed MESD(12-155) (FIG. 7D).

FIGS. 8A and 8B are photographs of western blots of the MESD in the Helacells that was treated with two different de-glycosylation enzymes:PNGase (FIG. 8A) and NAase (FIG. 8B), confirming that the upper bandswere glycosylated forms of MESD; and

FIG. 9A shows the flow chart of purification of LBD-apoER2. FIGS. 9B-9Dare photographs of far-western blots of purified LBD-apoER2 from Helacells using a His-Bind resin column, showing that purified LBD-apoER2binds to ApoE and RAP.

DESCRIPTION OF THE INVENTION

The present invention provides the QQ series of protein transductionreagents (QQ reagents) that can be used to deliver protein into anymammalian cells. In contrast to the currently available proteintransduction reagents, such as protein transduction domain (“PTB”) orcell penetrating peptide (“CPP”), the QQ reagents have an ability todeliver high concentrations of proteins into mammalian cells (up to50-200 μM). Utilization of QQ reagents is very simple, only requiringincubation of the protein of interest with the QQ reagent, passing theprotein solution through a quick-spin column to separate QQ reagentmodified protein from free QQ reagents and another incubation withcells. QQ reagents are safe to the cells since the majority of thecompositions of the QQ reagents are food additives that have been provedby FDA. More importantly, QQ reagents have two special features thatother currently available protein transduction reagents do not have: QQreagents can target different cell compartment and QQ reagents canprotect protein of interest from degradation by proteases.

In contrast to the other protein transduction reagents currentlyavailable, such as PTB or CPP, QQ reagents have the following featuresthat are unique and novel and distinctly different from the otherprotein transduction reagents:

-   (1) A non-invasive protein transduction method that is applicable to    any mammalian cells.-   (2) A high protein transduction efficiency method (50-200 μM protein    delivery into the cells).-   (3) A method that has the capability of targeting specific cell    compartments or cell organelles.-   (4) The QQ series reagents can either non-covalently or covalently    associate with protein (a unique feature) and dissociate from the    protein inside of cells. Thus, the protein under investigation is    not functionally altered.-   (5) The QQ series reagents are food additives that has been approved    by FDA, having no or little cellular toxicity.-   (6) The QQ series reagent protects proteins from protease    degradation inside the cells.-   (7) The application of QQ reagent is very simple and only requires a    step of incubation with cells. No any other step, such as molecular    cloning like CPP/PTB, is necessary.-   (8) The QQ series reagent enables multiple proteins to be delivered    into cells simultaneously or consecutively.

The reagents of the present invention can be used in numerousapplications, such as to: (1) utilize cell folding machinery to properlyfold bacterial expressed protein; (2) utilize cell post-translationalmodification machinery to properly post-translationally modifybacterially expressed proteins; (3) investigate the secretion pathwaysof the exogenous proteins that are delivered into the cell by the QQreagents; (4) investigate the degradation of the exogenous proteins thatare delivered into the cell by the QQ reagents; (5) studyprotein-protein interaction in living cells; and (6) study proteinfunction at a cellular level. The reagent can also be used for thefollowing: (1) Protein Therapy using the QQ reagent to target cellularcompartment; and (2) physiologically and pathologically relevant, atomicresolution cell biology.

The present invention demonstrates that the exogenous bacterialexpressed protein, after being delivered into mammalian cells, followsthe same secretion pathway as the endogenous proteins. This discoveryprovides a foundation for future applications of protein transductiontechnology, including protein therapy and atomic resolution cellbiology.

The present invention also provides a method that enables production oflarge quantity of native folded, post-translationally modified proteinsusing mammalian cell folding and post-translational modificationmachinery. The method uses bacteria to produce recombinant protein thatis then modified with the QQ reagent and delivered into mammalian cells.The mammalian cell machinery will fold and post-translationally modifythe transduced protein to produce native functional proteins that can bepurified from the cells with affinity column which can bind to the tag(His-tag or other tags) introduced into recombinant proteins. Theefficiency of recovery of recombinant protein from mammalian cell wasabout 30-60%.

The term “proteins” as used herein is intended to include any of a groupof complex organic macromolecules that contain carbon, hydrogen, oxygen,nitrogen, and usually sulfur and are composed of one or more chains ofamino acids. Proteins are fundamental components of all living cells andinclude many substances, such as enzymes, hormones, and antibodies thatare necessary for the proper functioning of an organism. Examples ofproteins tested include, but are not limited to: plasma protein (humanapolipoprotein AI, human apolipoprotein E and mouse apolipoprotein AI),ER residence protein (mouse MESD(1-195) and Receptor Associate Protein(RAP)), receptor protein (the ligand-binding domain of human apoEreceptor 2 (LBD-apoER2) and the YWTD β-propeller/EGF domains of LRP6,membrane protein mouse PMP22). These proteins have different hydrophobicand hydrophilic characters.

A “modified protein” is a protein that has been treated with the QQreagent of the present invention. The QQ reagent enables the protein tofunction normally while the protein is delivered into cells.

The term “folding” as used herein is the process whereby a proteinmolecule assumes its intricate three-dimensional structure. The processof folding in vivo often begins co-translationally, so that theN-terminus of the protein begins to fold while the C-terminal portion ofthe protein is still being synthesized by the ribosome.

The term “post-translational modification” as used herein is the processwhereby, after translation, a protein molecule get modified covalentlyat certain amino acids in several different ways, such as glycosylation,phosphorylation and ubiqutilization. Post-translational modificationsusually occur in the ER and the Golgi of the mammalian cells.

The term “cell machinery” as used herein is intended to mean themachinery within a cell that is used by that cell for protein foldingand for post-translational modification of the proteins produced by thatcell.

The method of the present invention includes the steps of expressing aprotein using bacteria, labeling the protein with probes, modificationof the protein with the QQ reagents, incubation of the modified proteinwith mammalian cells and protein folding and post-translationalmodification of the protein within cells. First, the protein isexpressed in bacteria. Optionally, the bacterial expressed protein canbe labeled with NMR and fluorescence probes. The bacterial expressedprotein is then modified using the QQ reagents of the present invention.The protein can then be delivered into mammalian cells by incubation ofthe QQ reagent modified protein with mammalian cells for several hours.The normal cell machinery can then properly fold andpost-translationally modify the transduced protein for production of thenative fold, post-translationally modified, functional proteins whichcan be purified using an affinity column.

The method enables analysis of the protein folding process in any cells,including both bacterial and mammalian cells. The method also enablesstudies of both protein structure and dynamics in the living cell, sincethis method allows one to label the protein in vitro and then transducethe labeled protein into mammalian cells for high-resolution biophysicalstudies, including in-cell NMR and in-cell fluorescence. Finally, themethod of the present invention enables a combination of atomicresolution structural biology techniques with cell biology techniques(KO/TG and NMR/fluorescence). The method can thus be used to studystructural biology at atomic resolution levels. In other words,structural biology can be studied at a physiologically andpathologically relevant level. By enabling such a study the methodallows for utilization of cell biology techniques, such as knockout,transgenic and knockdown techniques, along with high-resolutionstructural biology.

The method of the present invention can be used to test drugs todetermine how they affect protein folding. A “screen” can be used todetermine if the drug being used has potential problems with regard tocreating improper protein folding. As a screen, the method enablesproblems to be uncovered early in the developmental stages, to providebetter analysis with regard to potential side effects of a medication.

The ability of this system to deliver multiple proteins simultaneouslyor consecutively can be applied to test protein-protein interactions andmetabolic pathway interferences.

In-cell NMR and in-cell fluorescence techniques can also be developed tostudy protein structure/folding/trafficking/interactions within livingcells. The high transduction efficiency at 50-200 μM concentrationsinside cells makes in-cell NMR possible. In addition, the ability of QQreagents to target different cell compartments, where the reactionoccurs, ensures the physiological/pathological relevance of both in-cellNMR and in-cell fluorescence studies. As stated above, once the proteinhas been labeled, modified, and transferred into mammalian cells, theprotein can be monitored to identify relevant mechanisms, changes,trafficking and interactions. This is beneficial on a number of levels.First, it provides input as to the specific mechanisms involved withprotein and specifically protein folding. Second, it providesinformation with regard to the interactions surrounding proteins andprotein folding, since there are so many diseases that are related toimproper folding. It is critical to determine all cellular componentsthat are involved with the folding process. Third, it provides theability to visualize the actual 3D structure of the protein, oncefolded, inside cells.

The method of the present invention can also be used to delivertherapeutic proteins into tissues/cells for treatment of the diseasedtissue/cells using protein therapy. The special ability of targetingdifferent cell compartments by QQ reagents enables protein therapy tobecome a functional treatment option. Since the method enables proteinsto be modified, transferred into a cell and targeted at a specific cellcompartment, the protein can be used to alter the cell functions. Forexample, a modified protein can be transferred into a cell in need oftreatment and since the method of the present invention utilizes theexisting cellular folding machinery, the cell will fold the protein andthus the protein will be incorporated into the cell. As listed above,many diseases are known to be directly related to improper proteinfolding. For example, more than 50% of natural occurring mutants of LDLreceptor are so-called class 2 mutants that remain in the ER and Golgidue to either misfolding or partially folded. These mutants areassociated with Familiar Hypercholesterolemia (FH)—an autosomal dominantdisorder affecting about 1 in 500 individuals worldwide. Patients withhomozygotes develop severe atherosclerosis at a very early age.Knockdown of the LDLR mutation expression with supplement havingwild-type LDLR using QQ protein transduction technology may rescue thesepatients. Therefore, bypassing the existing disease-causing mutantproteins and adding a correct protein, diseases can be treated in a safeand effective manner.

Protein therapy is similar to gene therapy but instead of insertinggenes into the cells genome, proteins can be delivered into cells fortreatment. In a manner similar to gene therapy, protein therapy enablestargeted treatment of cells with specific proteins. For example, thereare numerous diseases that impact either protein production or themanner of protein folding. By inserting properly folded proteins intocells or tissues, the cells or tissues can be treated so that they areno longer in a disease state. Protein therapy has previously beenimpossible because proteases in blood and within the cells digest thedelivered proteins. In order for protein therapy to be practical, thedelivered protein has to be protected inside the cell from protosomes.The QQ reagent of the present invention protects the protein fromdegradation and thus enables the protein to be inserted into a cell aspart of a protein therapy.

The reagent of the present invention is a reagent that enables a proteinto be transferred into a mammalian cell. The reagent includes cationreagents, a lipid, and an enhancer. One example of an appropriate cationreagent is polyethylenimine (M.W.: 600 Da, 2,000 Da and 25,000 Da). Thelipid can be any lipid known to those of skill in the art to have thesame general properties as those listed herein. Examples of such lipidsinclude, but are not limited to, DOTAP, DOPE, POPC, and DMPE. Theenhancers can be any enhancer that significantly enhances cell loadingof cationized proteins. Examples of such enhancers in cell culturesinclude, but are not limited to MG132, protease inhibitor, CaCl₂, DMSO,growth factors and Na₂HCO. Other enhancers can also be used, including,but not limited to, cell membrane surfactants. The reagent can alsoinclude stabilizers and other inert carriers that do not affect thefunction of the reagent. As shown in Table 1 in the Examples, theconcentrations and specific compounds utilized can vary.

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided for thepurpose of illustration only, and are not intended to be limiting unlessotherwise specified. Thus, the invention should in no way be construedas being limited to the following examples, but rather, should beconstrued to encompass any and all variations which become evident as aresult of the teaching provided herein.

EXAMPLES

Methods:

General Methods in Molecular Biology:

Standard molecular biology techniques known in the art and notspecifically described are generally followed as in Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, New York (1989), and in Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Baltimore, Md. (1989) and inPerbal, A Practical Guide to Molecular Cloning, John Wiley & Sons, NewYork (1988), and in Watson et al., Recombinant DNA, Scientific AmericanBooks, New York and in Birren et al (eds) Genome Analysis: A LaboratoryManual Series, Vols. 1-4 Cold Spring Harbor Laboratory Press, New York(1998) and methodology as set forth in U.S. Pat. Nos. 4,666,828;4,683,202; 4,801,531; 5,192,659 and 5,272,057 and incorporated herein byreference. Polymerase chain reaction (PCR) was carried out generally asin PCR Protocols: A Guide To Methods And Applications, Academic Press,San Diego, Calif. (1990). In-situ (In-cell) PCR in combination with FlowCytometry can be used for detection of cells containing specific DNA andmRNA sequences (Testoni et al, 1996, Blood 87:3822.)

Some of the standard methods are described or referenced, e.g., inManiatis, et al. (1982) Molecular Cloning, A Laboratory Manual, ColdSpring Harbor Laboratory, Cold Spring Harbor Press; Sambrook, et al.(1989) Molecular Cloning: A Laboratory Manual, (2d ed.), vols 1-3, CSHPress, NY; Ausubel, et al., Biology, Greene Publishing Associates,Brooklyn, N.Y.; or Ausubel, et al. (1987 and Supplements) CurrentProtocols in Molecular Biology, Greene/Wiley, New York; Innis, et al.(eds.)(1990) PCR Protocols: A Guide to Methods and Applications AcademicPress, N.Y. Methods for protein purification include such methods asammonium sulfate precipitation, column chromatography, electrophoresis,centrifugation, crystallization, and others. See, e.g., Ausubel, et al.(1987 and periodic supplements); Deutscher (1990) “Guide to ProteinPurification” in Methods in Enzymology, vol. 182, and other volumes inthis series; and manufacturer's literature on use of proteinpurification products, e.g., Pharmacia, Piscataway, N.J., or Bio-Rad,Richmond, Calif. Combination with recombinant techniques allow fusion toappropriate segments, e.g., to a FLAG sequence or an equivalent whichcan be fused via a protease-removable sequence. See, e.g., Hochuli(1989) Chemische Industrie 12:69-70; Hochuli (1990) “Purification ofRecombinant Proteins with Metal Chelate Absorbent” in Setlow (ed.)Genetic Engineering, Principle and Methods 12:87-98, Plenum Press, N.Y.;and Crowe, et al. (1992) QIAexpress: The High Level Expression & ProteinPurification System QUIAGEN, Inc., Chatsworth, Calif.

FACS analyses are described in Melamed, et al. (1990) Flow Cytometry andSorting Wiley-Liss, Inc., New York, N.Y.; Shapiro (1988) Practical FlowCytometry Liss, New York, N.Y.; and Robinson, et al. (1993) Handbook ofFlow Cytometry Methods Wiley-Liss, New York, N.Y.

General Methods in Microbiology

Standard microbiology techniques known in the art and not specificallydescribed were generally followed as in Gerhardt et al. (Eds), Methodsfor General and Molecular Biology, American Society for Microbiology,Washington D.C. (1994), and in Woodford et al. (Eds), MolecularBacteriology: Protocols and Clinical Applications, Humana Press, Totowa,N.J. (1998) and in Demain et al. (Eds), Manual of IndustrialMicrobiology and Biotechnology, American Society for Microbiology,Washington D.C. (1986), and in Brock et al., Biology of Microorganisms,5.sup.th Edition, Prentice Hall, New Jersey (1988).

General Methods in Immunology:

Standard methods in immunology known in the art and not specificallydescribed are generally followed as in Stites et al. (eds), Basic andClinical Immunology (8th Edition), Appleton & Lange, Norwalk, Conn.(1994) and Mishell and Shiigi (eds), Selected Methods in CellularImmunology, W.H. Freeman and Co., New York (1980).

Example 1

Experimental Methods

Reagents:

Polyethylenimine (PEI) 600, 1200, 2,000 (2K), 8,000 (8K), 12,000 (12K),25,000 (25K), and 60,000 (60K) are purchased from Sigma-Aldrich. Lipids:DOTAP, DOPE, POPC and DMPE are purchased from Avanti polar lipids, Inc.

Enhancer: MG132 protease inhibitor cocktail are purchased fromSigma-Aldrich. DMSO, CaCl₂, growth factor and Na₂HCO are also purchasedfrom Sigma-Aldrich. The addition of these enhancers significantlyenhanced cell loading of cationized proteins.

Other enhancers can be used, including cell membrane surfactants. Thesematerials can be purchased from Fisher.

Protease inhibitor Cocktail is purchased from Sigma (Cat# P1860).

Antibodies:

Goat anti-apoE poly-Ab and mouse anti apoE mono-Ab are purchased fromChemicon international. Anti LRP8 mono-Ab is purchased from AbnovaCorporation. Goat anti 6×His-tag poly-Ab is purchased from InnovativeResearch. Anti RAP 7F1 is purchased from Innovative Research.

Anti goat IgG peroxidase conjugated is purchased from Sigma. Anti Rabbitand anti Mouse IgG peroxidase conjugated are purchased from Bio-Rad.

Cell Lines:

Hela cell line, GM 001300 cell line, BHK-570 cell line, CHO cell line,Raw cell line, MCF7 cell line, and HEK-293T cell line.

Recipes for QQ Series Reagents:

Stock Solutions:

Stock solutions of the following reagents are prepared as follows:

Cation Reagents: Stock Solution

600 1.2K 2K 8K 12K 25K 60K 50 mg/ml 20 mg/ml 50 mg/ml 20 mg/ml 20 mg/ml50 mg/ml 50 mg/mlwas dissolved in water and pH adjusted to 3.7-4.5 using HClLipids:

DOTAP DOPEPOPC DMPE 1 mg/ml 1 mg/ml 1 mg/ml 1 mg/mlEnhancers:

MG132 CaCl₂ Na₂HCO DMSO 1 mM 1M 1M 0.1M

TABLE 1 Recipes QQ Reagents^(a) 2K 8K 12K 25K 60K^(b) DOTAP DOPE POPCDMPE DMSO MG132 Ca²⁺ QQ1 200 μl — — — — 50 μl 50 μl — — 50 μl 5 μl 10 μlQQ2 200 μl — — 50 μl 5 μl 50 μl 50 μl — — 50 μl 5 μl 10 μl QQ3 100 μl100 μl  — 100 μl  — 50 μl 50 μl — — 50 μl 5 μl 10 μl QQ4 100 μl 50 μl 50μl 50 μl — 50 μl 50 μl — — 50 μl 5 μl 10 μl QQ5 100 μl 100 μl  — 100 μl — 50 μl 50 μl — — 50 μl 5 μl 10 μl QQ6 100 μl 50 μl 50 μl 50 μl — 50 μl50 μl — — 50 μl 5 μl 10 μl QQ7 100 μl 100 μl  50 μl 50 μl — 50 μl 50 μl— — 50 μl 5 μl 10 μl QQ8 100 μl 50 μl 50 μl 50 μl — — — 50 μl 50 μl 50μl 5 μl 10 μl QQ9 100 μl 50 μl 50 μl 50 μl — 25 μl 25 μl 25 μl 25 μl 50μl 5 μl 10 μl QQ10 100 μl 75 μl 50 μl 50 μl — 25 μl 25 μl 25 μl 25 μl 50μl 5 μl 10 μl ^(a)Total volume is 5 ml, based on 2-8 mg/ml proteinconcentration. ^(b)2K produces the least cellular toxicity whereas 60Kproduces the most cellular toxicity

For different experiments one can choose different combinations of QQreagents, e.g. to observe protein trafficking and location, QQ1 can beused which have the least cell toxicity. QQ1 can be used to incubateprotein for few days without causing significant cell death. For proteinfolding and post-translational modification, it requires maximum loadingin a short period of time, QQ8-QQ10 may be used, which have a highercell toxicity. However, cells survive with proper functions in a shortperiod of incubation time, such as 4-8 hours.

However, protein modified with 60K seems to give the best transferefficiency. Protein modified with 2K alone only give intermediatetransfer efficiency.

Protein Modifications with QQ reagents.

Proteins of interest are first dissolved into sodium phosphate buffer(pH7.0, NaCl 50 mM) at concentrations of 0.5-10 mg/ml, depends onprotein solubility. Protein solubility was found to influencecationization efficiency. To completely dissolve proteins, an overnightstir of the protein solution at room temperature is performed (with orwithout DTT at 3 mM for overnight, depending on if the protein ofinterest has Cysteine residues).

A lipid DOTAP/DOPE (1:1) emulsion was prepared using a method as thefollowing: 1 mg of DOTAP/DOPE (0.5 mg:0.5 mg=1:1) mixture was dissolvedin chloroform and dried under N₂ gas. The dried lipid film was thendissolved in PBS buffer, p1-17.0 and the lipid solution was sonicatedfor 3×30 seconds using a power of 7-8 on a sonicator from FisherScientific (Sonic Dismembrator, Model 100) with micro probe. The lipidsolution was further incubated at 37° C. for 2 hours until thesuspension becomes semi-clear. The prepared emulsion was store at 4° C.,which is stable for one month.

QQ series reagents (not included lipid emulsion, Ca and DMSO) were mixedin a tube, according to the recipe described above. The QQ reagent isthen titrated into the protein solution very slowly, drop by drop, whilestirring and then add the lipid emulsion. Once the addition of the QQreagents is completed, the protein solution is left at room temperaturefor 4 hours before use. During this period, a gently stir is necessaryto mix the QQ reagent with protein solution and also to allow theprotein modification reaction to complete. If precipitation is observed,the protein solution can be centrifuged at 14,000 rpm for 15 minutes toremove the precipitate. If the precipitate occurs, a BCA protein assaywill be carried out using the supernatant to check the amount of proteinremaining in solution. To ensure the efficiency of protein transfer intothe cells, the concentration of modified protein has to be high enoughat >0.5 mg/ml.

If the majority protein is precipitated, another QQ reagent can be usedfor protein modification. The QQ series reagents cover a wide range ofcationization reagents along with different lipids and enhancers, thusthe precipitation problem should be easily solved. The above procedurecan be repeated to prepare higher concentrations of modified proteinsolution for protein transfer into the mammalian cells.

The modified proteins are passed through a desalt column to separate themodified protein from remaining free QQ reagents. The purified proteinfractions can be concentrated using a spin column and are stable and canbe stored at 4° C. or −20° C. for between a few weeks to a few months.

Different QQ reagents can also be used for the best efficiency ofprotein transfer as well as the least cell toxicity. In addition,different proteins require modification with different QQ reagent forbest efficiency of protein transfer into cells. The following givesseveral examples:

-   -   For in vivo folding experiments of LBD-apoER2, QQ1 was used for        the best efficiency of protein transfer into the cells with        minimum cellular toxicity.    -   For in vivo glycosylation experiments of MESD, QQ1 was used for        the best efficiency of protein transfer into the cells with        minimum cellular toxicity.    -   For in-cell NMR experiment of apoE, QQ10 was used for the best        efficiency of protein transfer into the cells with a minimum        cellular toxicity.        Protein Loading into Mammalian Cells.

The fresh modified protein is mixed with cell culture medium (DMEM) with2% FBS or without, MG132 (3 ng/ml), protease inhibitor cocktail (2μg/ml), DMSO 30-50 ul/ml and growth factor (1 ng/ml). The newly preparedcell culture medium containing modified proteins is kept in a shakerotator for 10 minutes at room temperature to make sure that the proteinis completely dissolved. (For fluorescent imaging experiment, if anyprecipitation observed at this point, the protein can be centrifuged at13,000 for 5 minutes to remove the precipitation). The mammalian cellsare then added into this culture medium. Before adding the cells intothis medium, the cells are seeded for 2-3 days about 70-80% confluentfor monolayer cells for fluorescent imaging or 3-5 day old Helasuspending cells. The FBS concentration used in the experiments do notaffect the protein delivery.

For protein folding and glycosylation in living cells, both Hela cellsand lymphocytes GM001300 cells were used. Before loading modifiedproteins, the pre-conditioned cells are washed with DMEM medium. Thecells are then centrifuged at 1,000 rpm at 25° C. for 20 minutes. Thespin-packed cells are resuspended in DMEM cell culture medium and usedto load the modified proteins. For loading 4-6 mg bacterial expressedprotein, 2.0 ml spin-packed cells were used. The cells are suspendedinto 2 ml of the DMEM cell culture medium containing modified proteins(2 mg/ml) and then incubated at 37° C. for 2-5 hours.

For in-cell NMR experiments, Hela and lymphocytes GM001300 cells wereused. These cells are pre-treated using a medium that contains 10% D₂Oovernight. No cell morphology changes were observed after 10% D₂Otreatment, suggesting that cells can tolerate 10% D₂O without anyobvious cell toxicity. For loading 8 mg bacterial expressed protein, 1-2ml spin-packed cells were used. The cells are suspended into 2-3 ml ofthe DMEM cell culture medium containing modified proteins (4.0-8.0mg/ml) and 10% D₂O, and then incubated at 37° C. for 2.5 hours.

Once cell loading of the modified protein is started, the cells areclosely monitored using a microscope. Minor cell morphology changes wereobserved after loading the protein. However, these cell morphologychanges are reversible. Upon removing the loading medium and resuspendedcell into DMEM culture medium without modified protein for 5-10 minutes,cells are able to return to their original morphology. This minormorphological change of cells after loading with QQ reagent modifiedproteins depends on the amount of free QQ reagents in the cell culturemedium. Initially, a much larger amount of cell morphology changes weredetected in free QQ reagents that were not purified from the modifiedprotein. This is because that a large ratio of QQ reagent over proteinwas used and, after protein modification, a large amount free QQ reagentremained in the cell culture medium and caused cell morphologicalchanges. Once a purification step was added to remove free QQ reagents,only a minor cell morphology change was observed. However, thepurification step is generally only necessary when cellular toxicitybecomes an issue.

For both in-cell protein folding and glycosylation experiments, a timecourse study was performed and it was found that the modified proteinswere nearly completely transferred into the cells in 3 hours. This isdemonstrated by a western blot, showing that no protein remains in themedium after a 2 hour loading (FIGS. 2A and 2B). For in-cell NMRexperiments, the modified protein was loaded for 2-3 hours.

After loading the modified protein, the cells are washed with PBS buffer(pH7.4) for 3 times, 15 ml each time. This is a very important step forall experiments. The cells were carefully spun down at 500 rpm for 20minutes at 25° C., and add 15 ml PBS buffer to wash for 5 minutes with agentle re-suspension. The cells were again spin down and another 15 mlPBS was added to wash for 5 minutes. The third wash is a wash using alow pH PBS buffer (pH5) to remove any cell surface bound proteins. Thiswash is only for 1 minute, keeping the cells on ice, and quickly removesthe low pH PBS buffer by spin down the cells. The cells were thenresuspended into DMEM cell culture medium (pH7.4), which is ready forfurther experiments.

Protein Folding in the Living Cells:

The Ligand-Binding Domain of apoE receptor 2 (LBD-apoER2) is a294-residue protein that contains 42 cysteine residues, forming 21intra-molecular disulfide bonds. A proper folding of this protein isextremely difficult since it can form both intra- and intermoleculardisulfide bonds. Bacterial expression of this protein can't produce aproperly folded LBD-apoER2. In the mammalian cells, apoER2 is properlyfolded in the ER, with the help of folding enzymes and chaperones. Inparticular, there are two specialized chaperone proteins, RAP and MESD,specifically promoting the correct fold of the LDLR superfamily (1-2).RAP (Receptor Associated Protein) is a 323-residue ER-resident proteinthat specifically promotes the LBD fold, and escorts the mature,properly folded protein trafficking from the ER to the Golgi (3-4). Incontrast, MESD (Mesoderm development) is a 195-residue ER-residentprotein that contains an ER retention signal in its C-terminal domain(5-6). The biological function of MESD is to promote a proper folding ofthe YWTD/EGF domain of the LDLR superfamily (5-7).

LBD-apoER2 was used as a model protein for this experiment, simplybecause it is very difficult to correctly refold in vitro due to itsrich in cysteines. The present invention utilizes the folding machineryof the mammalian cells to help LBD-apoER2 proper folding. Based on this,the present invention provides transferring bacterial expressedLBD-apoER2, which is not folded properly and non-functional in terms ofligand-binding, into the mammalian cells. After being transferred intothe cells, the misfolded LBD-apoER2 has an ability of entering cellapparatus, such as ER, and therefore can be properly folded in the ER,similar to the endogenous apoER2. This is supported by several previouspublications, indicating that the transferred exogenous proteins areable to reach inside of nucleus of the mammalian cells (7-8). Thefolding machinery in the ER of the mammalian cells makes the bacterialexpressed protein fold properly.

LBD-apoER2 is expressed in bacteria as a His-tag fusion protein.Bacterial expression was optimized and is able to produce 300 mgLBD-apoER2 from one-liter cell culture. Using SDS-PAGE, it was shownthat the bacterial expressed LBD-apoER2 is mis-folded and form differentoligomers due to inter-molecular disulfide bond formation. Using aligand blot (far western), it was demonstrated that the bacterialexpressed LBD-apoER2 is incapable of binding to its ligand, such as RAPand apoE.

QQ series reagents were used to modify LBD-apoER2. In this case, QQ1 isused to modified LBD-apoER2, following the protocol describe above.After modification, LBD-apoER2 (1.0 mg/ml) is incubated with Hela cellsat 37° C. for 2 hours to allow cells to load the modified LBD-apoER2.The cells were gently spun down and a western blot of the medium was runusing an anti-his-tag antibody to check the loading efficiency. In mostcases, the cells loaded most of the modified LBD-apoER2, and westernblot of the medium either show no band or a very weak band ofLBD-apoER2. The cell pellet is resuspended into DMEM cell culture mediumand incubated for a period that allows the mis-folded LBD-apoER2 to foldinto its native conformation in living cells.

The incubation is stopped by spin-down of the cells, which are thenwashed. The cell pellet is lysed using a sonication method with additionof protease inhibitor. The sonication uses a power of 7-8 on a sonicatorfrom Fisher Scientific (Sonic Dismembrator, Model 100) with a microprobe. Each sonication lasts 1 minute on ice and is repeated threetimes. There is a 3 minutes interval between sonications to prevent overheating. The supernatant fractions are combined. The pellet is dissolvedinto a buffer that contains 1% SDS. Both supernatant and pelletfractions are used for ligand blots (far western blots) or pull-downexperiments using RAP and apoE as the ligand, to probe the folding andfunction of LBD-apoER2

Two assays are used to probe the proper folding and functions of the incell refolded LBD-apoER2. One is a ligand-blotting assay and the otheris a pull-down assay. The details of these two assays are described inthe following:

Ligand Blot:

Cell lysate (supernatant or pellet fraction) is loaded onto either anon-reducing SDS-PAGE (For RAP) or a native gel (apoE/DMPC). The gel isrun in a cold room. The protein is then transferred onto anitrocellulose membrane at 380 mA for 2 hours. After transfer, themembrane was incubated at room temperature with either RAP (40 μg/ml in2% dry milk PBS) for 2 hours or incubated with apoE/POPC complex (50μg/ml in 2% dry milk PBS) for 2 hours. The membrane is then washed threetimes with 20 ml/each PBS for 5 minutes each time, to remove unbound RAPor apoE/POPC. The membrane is incubated with anti-RAP mAb or anti-apoEpoly Ab for 1 hour in 2% dry milk PBS, followed by incubation withHPR-conjugated secondary Ab for 1 hour. Finally, the membrane isdetected using ECL (Pierce). Using this assay, a band is observed at themolecular weight of LBD-apoER2 (34 kDa) for the complex ofligand/LBD-apoER2.

Pull-Down:

Cell lysate (supernatant or pellet fraction) is in the binding buffer (2mM imidazole) for the His-tag beads and loaded onto the beads. Since theLBD-apoER2 contains a his-tag, this protein binds to the his-tag beads,whereas all other proteins in the cells do not bind to the beads and canbe washed out with binding buffer. After loading, the beads (˜0.2 ml) iswashed with 5×5 ml washing buffer, containing 10 mM imidazole, to removethe unbound proteins from the His-tag beads. The purified beads are thenincubated for 1 hour with either RAP or apoE/POPC particles that areprepared previously using the cholate dialysis method. This step allowsthe refolded LBD-apoER2 to bind to either RAP or apoE/POPC. The beadsare then washed with 5×5 ml PBS buffer, pH7.4, to remove the unboundligand. The ligand/LBD-apoER2 complex is eluted out using an elutionbuffer containing 0.2 M immidizole. An aliquot of the elutedligand/LBD-apoER2 was mixed with 2% SDS loading buffer (for SDS-PAGEonly) and loaded on a SDS-PAGE or native gel, and is subjected towestern blots using the anti-ligand antibodies. Using this assay, a bandshould be observed at the ligand molecular weight (SDS-PAGE) or ligandand LBD-apoER2 complex (Native Gel), if LBD-apoER2 is functional andbinds to the ligands. For example, in RAP/LBD-apoER2 pull-downexperiment should show a band at 73 kDa.

For both ligand blotting and pull-down experiments, several controls areincluded:

-   1. Hela cell lysate only (without loading the modified protein, to    test endogenous protein).-   2. Bacterial expressed LBD-apoER2 that is not properly folded (serve    as a negative control).-   3. ApoE/POPC particles (Serve as a control).-   4. Cell lysate from unload protein cells as column wash control.    In a pull-down experiment, all of the control groups were negative.    Protein Post-Translational Modifications in Living Cells:

Another question is whether the transferred exogenous proteins undergopost-translational modifications inside of the cells. These exogenousproteins may undergo post-translational modifications inside the cellsif they are able to get into the ER and Golgi. Our data indicated thatthe transferred exogenous protein, LBD-apoER2, is able to get into theER for its proper folding.

Protein glycosylation is the important step in studying proteinpost-translational modifications. First, it must be confirmed whetherthe transferred exogenous proteins undergo glycosylation. Once this isproven, testing on phosphorylation and ubiquitination of the transferredexogenous proteins can occur.

MESD, a known glyco-protein, have been transferred into the cells. Thecells were lysised and the proteins were probed using western blots.Higher molecular weight bands were observed for MESD than the band ofbacterial expressed MESD as controls. In addition, the molecular weightof MESD band increases along with the length of incubation of the cellsafter protein transformation. For example, a one-hour incubation aftertransformation resulted in a two bands at molecular weight of 25 and 35kDa (MESD is a 25 kDa protein). A five hour incubation generated anadditional band at ˜45 kDa for MESD. The experiment was repeated severaltimes and the same phenomena were always observed. The increase inmolecular weight of MESD is due to glycosylation, since deglycosylationenzymes converted the two higher molecular weight bands (35 and 45 kDa)into one single band at 25 kDa that is the same as the bacterialexpressed MESD.

Experimental Details:

0.5 mg of modified MESD is incubated with Hela or GM001300 cells at 37°C. for 1-5 hours. The cell culture medium (5 ml) contains 5% FBS, 1ng/ml of MG 132 and 2 μg/ml protease inhibitor cocktail. Afterincubation, cells were washed with 3×15 ml PBS. Each wash includesadding 15 ml PBS into the cell pellets and re-suspension, following byspin-down the cells at 500 rpm for 20 minutes at 25° C. The cells arelysed using sonication and spun down at 6000 rpm for 5 minutes. Thepellet is dissolved using a SDS loading buffer (2% SDS) and heat at 80°C. for 15 minutes with DTT. Both the supernatant and pellet fractionsare probed with a western blot using a polyclonal antibody against MESD.At 1 hour cell loading, the western blots of both supernatant and pelletfractions show mainly a band at a molecular weight of 25 kDa, which isidentical to the band of bacterial expressed MESD, with a minor band at35 kDa. However, at 5 hour cell loading, an additional band at 45 kDawas observed.

Enzymatic de-glycosylation reaction of the products was carried outusing neuraminidase and PNGase. Time courses of both enzymatic reactionsfinally converts both the 35 and 45 kDa bands to the 25 kDa band whichis identical to the band of the bacterial expressed MESD. Thus, thisresult confirms that MESD is glycosylated inside the cells. MESDcontains an ER retention signal in its C-terminal domain. Once thisprotein gets inside the ER, it may stay inside the ER, therefore, issubjected to post-translational modification, including glycosylation.

Exogenous MESD Follows the Same Secretion Pathway as Endogenous MESDInside Hela Cells

Since MESD is glycosylated inside Hela cells, it suggests that thetransduced MESD travels to the ER and Golgi, the two cell compartmentswhere post-translational modifications occur. To confirm this, anexperiment was performed to identify the intracellular location of MESDafter transduction. First, MESD was labeled with green-ArrayIt and thenpurified from free green-Arraylt using a desalt column. Fluorescencelabeled MESD was then modified with QQ1. The modified, fluorescencelabeled MESD was incubated with Hela cells for 3 hours and then thecells were taken for fluorescence imaging using a ApoTom (Zeiss)Ax10plan 2 Imaging system.

The fluorescence imaging clearly showed that the MESD was primarilylocated in the peri-nuclei areas that are either the ER or the Golgi.This data confirmed that the exogenous MESD traveled to the ER and theGolgi where the folding and post-translational modification occurred. Itseemed that the QQ reagent could target MESD into the ER and the Golgi.In addition, the data further suggested that the exogenous MESD stayedin the ER and the Golgi. To further identify what caused MESD to travelto and stay in the ER and the Golgi, a MESD construct, MESD(12-155), wasprepared that removed the ER retention signal. The data indicated thatno glycosylation was observed in this case (FIGS. 5A-5B). Therefore, thedata indicated that it was the REDL ER retention signal of MESD thatdirected the exogenous MESD to travel to and stay in the ER and theGolgi after transduction inside the cells. Thus, the exogenous MESDfollows the same secretion pathway as the endogenous MESD.

It is a very important conclusion, since this conclusion indicates thatthe exogenous proteins follow the Blobel's “Signal Theory”, suggestingthat the signal sequences of proteins direct the fate of all proteins,regardless their endogenous or exogenous origins, once they are insidethe cell. This conclusion further indicates that the exogenous proteinsfollow the same secretion pathway as that of endogenous proteins,providing the physiological and pathological relevance of theapplications of protein transduction technology.

Experimental Details:

2 mg MESD in 200 μl PBS buffer, pH 7.4 (10 mg/ml), was dissolvedovernight. The solution was spun for 10 minutes at 12,000 rpm at roomtemperature. 70 μl protein solution was taken and 0.5 μl ofgreen-Arraylt was added at room temperature. An incubation was carriedout for 6 hrs to overnight in cold room and then purified using a desaltspin-column at 10,000 rpm for 2 minutes to remove free dye. Thepurified, fluorescence labeled protein was modified by QQ1, asdescribed, using 10 μl of QQ1 stock solution, and incubated overnightwithout further purification.

Hela cells were seeded three days before the experiment. Hela cells wereincubated with QQ1 modified, fluorescence labeled MESD in DMEM culturemedium for 1-3 hours in 37° C. The cells were very healthy after theincubation, displaying normal morphology. The cells were washed with PBSseven times to remove any MESD in the medium. Hela cells were then usedfor fluorescence imaging using an ApoTom (Zeiss) Ax10plan 2 Imagingsystem. The fluorescence imaging experiments were carried out at 486 nmwith dipping lens in the PBS with anti-fading reagents and proteaseinhibitor to observe live cells. Photos were taken under light andfluorescent overlayers to view the cell body and fluorescence labeledMESD.

High-Level Production of the Properly Folded, Functional Protein Usingin-Cell Folding Technology

Using an in-cell folding technology, it was demonstrated that mammaliancell folding machinery could be used to properly fold LBD-apoER2, whichwas impossible to fold in bacteria (Section VII). The next goal was topurify this properly folded, function LBD-apoER2 for structural andfunctional studies. This technology can be used to produce largequantities of properly folded, functional LBD-apoER2. Since thebacterial expression system produces 300 mg/liter purified. LBD-apoER2,the technology can produce hundred mg quantity of the properly folded,functional LBD-apoER2. This is a major advance in production offunctional proteins, which is extremely important for pharmaceuticalindustry, since the technology can produce hundred milligrams oftherapeutic protein for disease treatment purpose.

Since a his-tag was introduced in the N-terminal domain of LBD-apoER2,protein purification of in-cell folded LBD-apoER2 was carried out used ahis-tag binding resin column. The results indicated that from 1.5 mgbacterial expressed LBD-apoER2, 0.5-0.75 mg properly folded, functionalLBD-apoER2 can be obtained. This purification was repeated several timesand obtained a similar yield. It was also demonstrated that the purifiedprotein is properly folded and biologically functional and is able tobind to both RAP and apoE/POPC particles.

Experimental Details:

The first part (Protein loading and in-cell folding) is essentially thesame as above. The only difference is the incubation time after proteinloading into the cells. The cells were loaded with modified LBD-apoER2for 3 hours. After loading, the cells were incubated in the DMEM cellculture medium, containing 5% FBS, MG132 (3 ng/ml), protease inhibitorcocktail (2 μg/ml), for 2-5 hour before spin-down, to ensure that mosttransferred LBD-apoER2 is correctly folded.

After incubation, cells were washed using PBS three times, 15 ml eachtime. After wash, the cells were spun-down gently at 2000 rpm for 5minutes. The cell pellet (0.5 ml) was resuspended in 2 ml cell lysisbuffer, which is a PBS buffer, containing 2% Triton-X, 2 μM PMSF, 2μg/ml protease inhibitor and 2 ng/ml MG132, pH 7.4. The cell suspensionwas incubated on ice for 30 minutes and sonicated for 30 seconds at asonication power at 4 using a sonicator from Fisher Scientific (SonicDismembrator, Model 100) with micro probe. Cell lysate was spun down.The cell pellet was dissolved again using cell lysis buffer andsonicated again for 30 seconds. The supernatant fractions were combinedand loaded on a small his-tag binding column (0.3-0.5 ml). The columnwas washed using 50 ml PBS buffer first and then washed again with 25 mlPBS buffer containing 2 mM imidazole. A 20 μl of his-tag beads weretaken out and dissolved into 20 μl of SDS loading buffer. This samplewas loaded onto a SDS-PAGE to check the protein purity. If the proteinis not pure, more washing buffer would be used. This purification wascarried on in the cold room. All buffers, including loading buffer andwashing buffer, contain 2 μM PMSF and 1 ng/ml MG132. Protein was elutedwith 1 ml elution buffer that contains 300 mM imidazole, 50 mM phosphatebuffer, containing 50 mM NaCl, 2 μM PMSF and 1 ng/ml MG132.

The eluted protein was dialyzed overnight against 150 ml PBS buffer at4° C. using a small dialysis cassette (Maximum volume: 3 ml). The PBSbuffer was changed twice during dialysis to ensure the removal ofimidazole. After dialysis, the properly folded LBD-apoER2 was taken outof the dialysis cassette, add MG132 at 1 ng/ml and store at −80° C.freezer. For long-term storage, the protein sample was freeze-dried intopowder.

The purified LBD-apoER2 was probed for its folding and function. Twofunctional assays: Ligand blot and pull down experiments were performedto probe the function of LBD-apoER2. In addition, both RAP and apoE/POPCwere used as the ligand for LBD-apoER2. Both assays demonstrated thatthe purified LBD-apoER2 is functional and capable to bind to both RAPand apoE/POPC, whereas the bacterial expressed LBD-apoER2 was notfunctional.

In addition to LBD-apoER2, the present invention enables one to prepareproperly folded YWTD/EGF domain of apoER2. For this purpose, the firstYWTD/EGF domain and the first two YWTD/EGF domains of LRP6 werebacterially expressed. LRP6 is an important cancer suppressor thatcontains four YWTD/EGF domains before the three LBD repeats andtransmembrane and cytoplasmic domains. This receptor is coupled with thewnt signaling, serving as a co-receptor for wnt, which is critical tomany human diseases, including cancer and Alzheimer's disease. LRP6 andapoER2 have a complete different modular structure arrangement for theLBD and YWTD/EGF domains. In addition, production of the followingfunctional proteins are currently carried out: LCAT, CETP, PLTP andseveral membrane proteins, such as ABC-G1, ABC-G4, and SR-BI. Theseproteins are important proteins that are involved in reverse cholesteroltransport pathway and responsible for enhancement of the HDL (Goodcholesterol) level in plasma. In addition, mouse PMP22 has also beenexpressed using E. coli. PMP22 is a membrane protein that containsputative four transmembrane domains. The preliminary data indicates thatbacterial expression of these proteins produces an incorrect folding,thus are not functional.

In-Cell Nuclear Magnetic Resonances (NMR) Technique

Current high-resolution structural biology techniques, including X-raycrystallography and NMR, allow for structural determination of protein,DNA and RNA at an atomic resolution. However, these techniques onlydetermine the structures of macromolecules in the test tube. There is nohigh-resolution structural biology techniques permits to solvestructures of these macromolecules in the living cells.

Several studies, using NMR, investigate protein structures in the livingcells, primarily focusing on bacterial cells and oocytes (1-4). For NMRstudies, a protein has to be isotropically labeled with stable isotopes,such as ¹⁵N and ¹³C. For large proteins (MW>25 kDa), protein has to betriple-labeled with ²H, ¹⁵N and ¹³C. For bacterial cells, a protein canbe high-level expressed in minimum medium (HN₄Cl, nitrogen source,glucose: carbon source) and isotope labeling can be readily performedwith a low cost (using ¹⁵NH₄Cl as nitrogen source, ¹³C-glucose as carbonsource and D₂O). However, this isotope labeling strategy for bacterialexpression not only labels the protein of interest, but also labels allthe other bacterial proteins. Since the protein of interest ishigh-level expressed at >10-100 folds higher than bacterial proteins,the NMR signals are mainly come from the protein of interest.Nevertheless, bacterial proteins will give rise to a high backgroundsignals in the NMR spectra. In contrast, oocytes are large cells and canbe used to micro-inject a large amount of isotope labeled protein intothe cells. Thus NMR data of the oocytes can be collected. These studiesutilized techniques that can only apply to bacterial cells and oocytes.Currently, there is no technique that permits to study protein structureat atomic resolution in the mammalian cells.

Using the present technique, isotropically labeled proteins can beproduced using bacteria. The isotropically labeled proteins are modifiedusing QQ reagents and then incubated with the living mammalian cells.The modified, isotropically labeled proteins can be transferred intothese mammalian cells, which allow collection of 1D/2D/3D-NMR data forstructural determination purpose. In order to collect 2D/3D NMR data forstructural determination of a protein in the living cells, several majorproblems have to be solved:

-   -   1. Higher protein concentrations in living cells. NMR technique        is not a sensitive technique that requires a high sample        concentration for detection for 2D/3D NMR experiments. With a        cold probe of 600 MHz NMR instrument, a sample of 50-200 μM is        required for a 1-2 hour experiment for a regular 2D HSQC        experiment. In contrast, for a 3D NMR experiment, a sample of        300-500 μM is required for a 2-3 day experiment.    -   2. Cell survival for at least for 3 days as a cell slurry state        in the NMR tube. Since a 3D-NMR experiment requires 2-3 days        data collection time, the cells have to be survival for at least        3-days as a slurry state in the NMR tube.    -   3. Cells have to be trained in a cell culture medium with 5-10%        D₂O. Since NMR experiment requires 5-10% D₂O to lock the        magnetic field, the cells have to be able to survive in a        culture medium that contains 5-10% D₂O.    -   4. Subcellular protein locations. To ensure physiological and        pathological relevance of in-cell NMR, the transduced protein        has to be targeted to the correct cell compartments where the        biochemical reaction occurs for this protein.        Experimental Details:

Proteins were modified based on the method described above with QQ9 orQQ10. For each in-cell NMR experiment, 4-10 mg of protein was modifiedfreshly each time. If there is any precipitate after proteinmodification, the modified protein solution was spun down andsupernatant was used for further experiments. The QQ reagent modifiedprotein was purified from free QQ reagents using a desalt spin column.The purified protein was concentrated to have a high concentration thatensure >1-2 mg/ml after mixing with the cell loading medium thatcontains: 0.5% FBS, 10% D₂O, MG132 1 μM and protease inhibitor 2 μM.

The cells (Hela or GM001300) were pre-incubated with 10% D₂O in the DMEMcell culture medium with 10% FBS for 24 hours. The cells were then mixedwith the cell-loading medium. After mixing with the loading medium, thecells were closely monitored every ten minutes using a microscope.Several different QQ reagents were tested and it was found thatdifferent QQ reagents produced different cell toxicity. The generalstrategy is to choose a QQ reagent that gives a good cell loadingefficiency of the modified protein while produces the least celltoxicity. This is because the cell preferably survives for more thanthree days in the NMR tube for NMR data collection. QQ6-QQ10 reagentsgenerally serve for this purpose.

Protein was loaded into cells for about 2-3 hours at 37° C., on arotator depending on cell morphology changes. If there was no or aminimum cell morphology change, cells could be loaded for a longer time,such as 2-4 hours, so that more protein could get into the cells. Incontrast, if there was a significant cellular morphology change or cellsstarted to lysis, the loading was stopped. Cells were scraped fromflasks and cell number in the suspension was adjusted into 1×10⁹/ml.Total volume of cell suspension used in NMR experiment was 0.5-1.0 ml(packed volume). A time course of protein uptake was tested for eachindividual protein, to identify the best loading time with minimum celltoxicity and maximum protein loading. After incubation, cells werecentrifuge gently at 500 rpm for 20 minutes and washed with 4×5 ml ofDMEM. Each wash followed by gentle spin to remove the wash medium. Afterwashing, the cells were resuspended into 1 ml of NMR sample buffer.

NMR sample buffer contains (1 ml): PBS 0.65 ml, DMEM 0.2 ml, 0.1 ml D₂O,0.05 ml of 4% BSA, MG132 3 protease inhibitor 2 μl, antibiotics mixture10 μl and cell culture vitamin 100× solution 10 μl. This step iscritical for good NMR experiments, which should be very careful to makecell slurry as homogenous as possible to avoid any macroscopic clumps ofcells. Any macroscopic clumps have to be removed before the NMRexperiments. The cells were carefully transferred into a 5 mm NMR tubeand let the cells settled for 10-30 minutes.

After NMR experiments, the cells were taken out from the NMR tube andthe tube was washed with 2×3 ml NMR sample buffer. The cells were thenused for several assays to test cell viability after NMR experiments.The results indicated the following results:

Cell viability was evaluated by MTT assay and trypan blue stain assay.For human apolipoprotein A-I loaded into Hela cells, MTT assay showed70% cell viability for NMR sample after 24 hour NMR experiment at 30°C., as compared with the control that is the cells in the culture dish.The trypan blue stain assay indicated that >90% of cells were alive. Forhuman apolipoprotein E loaded into Hela cells, the trypan blue stainassay indicated that >75% of cells were alive after 3-day NMRexperiments at 30° C.

All NMR experiments were collected using a Varian 600 MHz NMRspectrometer with a cold probe. Primarily, ¹H-¹⁵N HSQC experiments ofseveral different proteins were collected, including humanapolipoprotein AI (243-residues), human apolipoprotein E (299-residues)and mouse MESD (195-residues). These proteins are large proteins andtriple-labeled protein samples were used.

Throughout this application, author and year, and patents, by number,reference various publications, including United States patents. Fullcitations for the publications are listed below. The disclosures ofthese publications and patents in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

The invention has been described in an illustrative manner, and it is tobe understood that the terminology that has been used is intended to bein the nature of words of description rather than of limitation.

Obviously, many modifications and variations of the present inventionare possible in light of the above teachings. It is, therefore, to beunderstood that within the scope of the described invention, theinvention may be practiced otherwise than as specifically described.

The invention claimed is:
 1. A method of protein transduction intomammalian cells, the method comprising the steps of: modifying a proteinto be introduced into a cell with a reagent that enables the protein tobe efficiently delivered into mammalian cells, the reagent comprising: acation reagent and a lipid in a carrier, wherein the cation reagentcomprises polyethylenimine; thereby producing a non-covalently bound,dissociable complex of: the protein to be introduced into a cell, thecation reagent and the lipid; and delivering the non-covalently bound,dissociable complex into mammalian cells.
 2. The method according toclaim 1, further including the step of expressing the protein inbacteria prior to said modifying step.
 3. The method according to claim2, further including the step of in vivo labeling the proteins with theNMR-active isotopes during bacterial expression.
 4. The method accordingto claim 1, further including the step of in vitro labeling the proteinwith small molecule fluorescence probes prior to modifying the protein.5. The method according to claim 3, further including monitoring proteinactivity.
 6. A composition for transduction of a protein into a cell,said composition comprising: a non-covalently bound, dissociable complexof: a protein to be introduced into a cell; a cation reagent, whereinthe cation reagent comprises polyethylenimine; and a lipid in a carrier.7. The composition according to claim 6, wherein said lipid is selectedfrom the group consisting essentially of DOTAP, DOPE, POPC, and DMPE. 8.The composition according to claim 6, wherein said enhancer is anenhancer that enhances cell loading of the dissociable complex.
 9. Thecomposition according to claim 8, wherein said enhancer selected fromthe group consisting essentially of DMSO, MG132, CaCl₂ and growthfactor.
 10. The composition according to claim 6, further including atleast one selected from the group consisting essentially of cellmembrane surfactants, stabilizers, and other inert carriers.
 11. Amethod of specific delivery of an exogenous protein into a targetsub-cellular compartment, the method comprising the steps of: modifyingthe exogenous protein with a composition according to claim 6, producinga non-covalently bound, dissociable complex of: the exogenous protein; acation reagent; and a lipid in a carrier, wherein the cation reagentcomprises polyethylenimine; and delivering the non-covalently bound,dissociable complex into mammalian cells.
 12. A method of treating aprotein to enable proper in cell folding by exposing the protein to thecomposition of claim 6 for enabling utilization of cell foldingmachinery of the mammalian cells.
 13. A method of treating a protein toenable proper in cell post-translational modification by exposing theprotein to the composition of claim 6 for enabling the utilization ofcell post-translational modification machinery within mammalian cells.14. A method of analyzing secretion pathways of exogenous proteins afterbeing transduced into mammalian cells using fluorescence imaging by:forming a non-covalently bound, dissociable complex of the exogenousprotein according to claim 6; delivering the protein into mammaliancells; and analyzing the secretion pathway of the protein usingfluorescence imaging.